The Role And Mechanism Of LncRNA TUG1 In The Treatment Of Spinal Cord Injury With Photobiomodulation

Background:Spinal cord injury(SCI)is a refractory disease caused by trauma,which is caused by movement below the injury plane and loss of sensory function.At present,there is no effective treatment.There are two stages to the pathological process after severe spinal cord injury: primary and secondary.Primary injury is a local compression injury of the spinal cord caused by the initial mechanical impact,which is usually irreversible.Secondary injury continues from primary injury.Secondary inflammatory reaction is an important pathological mechanism of SCI,and regulating inflammatory reaction is one of the strategies of SCI treatment.Bone marrow-derived macrophages(BMDM)are the main cells involved in the secondary inflammatory reaction.They are recruited to the injured area after injury.Their M1 type polarization can promote the inflammatory reaction and neuronal apoptosis,further aggravating the progress of SCI.Therefore,in-depth study of the mechanism of inflammatory response induced by bone marrow-derived macrophages in secondary SCI is crucial to promote the repair of SCI.Photobiomodulation(PBM)is a classical physical therapy method with significant antiinflammatory and tissue repair effects.In many clinical settings,it has gained popularity because of its good controllability,small trauma and lack of toxic side effects and side effects,such as skin and nerve injury.Different nanometers of low-intensity laser have different wavelength functions,and play an important role in regulating inflammation,wound healing,nerve regeneration,osteogenesis and other aspects.Our team previously verified the safety of PBM treatment in the piglet model,and found that PBM can inhibit the polarization of M1 phenotype of BMDM and reduce inflammation.However,the safety and effectiveness of clinical application of PBM and the potential target and mechanism of its biological function are still unclear.Long non-coding RNA(lncRNA)is abnormally expressed after SCI,and can participate in the pathological processes,such as apoptosis,inflammatory reaction,angiogenesis,nerve repair and scar formation.However,little is known about the potential link between lncRNA and PBM.Therefore,this study aims to further carry out prospective research on clinical treatment of PBM,and further explore the specific mechanism of PBM inhibiting inflammatory reaction and promoting SCI repair.This is conducive to the clinical promotion of PBM treatment and provides theoretical support for clinical application.Methods:In the clinical study,we included 27 SCI patients(SCI group=12,PBM group=15)who met the research criteria.After admission,the patients were given PBM treatment for 7consecutive days.The safety of clinical treatment of PBM was evaluated by vital signs,infection indicators,photosensitive indicators and coagulation related indicators.The peripheral serum of patients in SCI group and PBM treatment group was collected,and the effect of PBM treatment on the expression of inflammatory factors was detected by ELISA.The patients were followed up for three months after operation,and the recovery of motor and sensory functions in SCI group and PBM treatment group were evaluated by ASIA score.In the part of basic research,we first extracted and induced primary bone marrowderived macrophages in vitro.The extracted and induced macrophages were identified by immunofluorescence assay,RT-PCR,western blot and flow cytometry.The cells were further irradiated with PBM,and the differential lncRNA before and after PBM irradiation was screened by transcriptional sequencing analysis and RT-PCR.The regulatory effects of lncRNA TUG1 on the polarization of BMDM,inflammatory response and DRG axon growth were explored by adenovirus transfection,western blot,RT-PCR and immunofluorescence.We performed bioinformatics analysis and screening,as well as dualluciferase reporter assay,nucleocytoplasmic separation experiment and the above molecular biology experiments,it is verified that the lncRNA TUG1-miR-1192/TLR3 axis can be used as the specific mechanism of PBM.We randomly divided male C57BL/6J mice into three groups,namely sham operation group(Sham group),SCI group and SCI+PBM treatment group,and established mice SCI model and PBM irradiation model.The mice in the PBM treatment group were treated with continuous irradiation for 28 days.Fluorescence in situ hybridization,RT-PCR,western blot and immunofluorescence assay were used to detect the effects of PBM treatment in vivo on lncRNA TUG1,TLR3,inflammatory cytokines and neuronal survival.Basso Mouse Scale(BMS)score,Louisville Swim Scale(LSS)score scale and footprint test were used to evaluate the recovery of motor function in mice.Results:Clinical studies showed that there were no significant changes in body temperature,heart rate,blood pressure,respiration and oxygen saturation index before and after PBM treatment.The infection index(White blood cell,Neutrophils,Hypersensitive C-reactive protein,Procalcitonin),photosensitivity index(eosinophil,basophil)and coagulation function index(prothrombin time,activated partial thrombin time,thrombin time)after irradiation were the same as those before.TNF-α,IL-1α,IL-1β,and IL-6 were significantly reduced in the PBM treatment group compared to SCI.The three-month follow-up of patients in the PBM treatment group showed that the score of sensory function was significantly improved.At the cellular level,following PBM irradiation,M1 macrophages expressed more lncRNA TUG1 than after M1 macrophages were not irradiated.After transfected TUG1 knockout and adenovirus overexpression,western blots showed that knockdown TUG1 could reduce the expression of i NOS(M1 phenotype marker)in M1 macrophages,and the expression of i NOS was further reduced after PBM treatment.Overexpression of TUG1 promoted the expression of i NOS in M1 macrophages,and PBM treatment could reduce the increase of i NOS expression.In addition,inflammatory cytokines(TNF-α,IL-1α,IL-1β,IL-6 and CXCL2)expression was consistent with i NOS.Liquid transfer test showed that the knockdown of lncRNA TUG1 promoted the axon length of DRG,while the overexpression of lncRNA TUG1 had the opposite effect.In terms of mechanism,bioinformatics analysis shows that TLR3 can be used as the core gene in the lncRNA TUG1-miRNA-m RNA pathway.In addition,lncRNA TUG1 and TLR3 can bind to miR-1192 respectively.Rescue experiment showed that TUG1 could regulate the expression of TLR3 by sponging miR-1192.Knockout of TLR3 can inhibit the expression of i NOS,p-NF-κB and five inflammatory cytokines,while over-expression of TLR3 has the opposite effect.In vivo experiment showed that the expression of lncRNA TUG1,TLR3,TNF-α,IL-1α,IL-1β,IL-6 and CXCL2 were the highest 7 days after SCI,and decreased after PBM treatment.Compared with the injured group,the number of neurons in the injured area was increased 7 days,14 days and 28 days after PBM treatment.In addition,the scores of BMS and LSS of mice in the PBM treatment group were significantly improved,and had statistical significance from the 7th day.Footprint test showed that PBM treatment promoted the step size of mice.Conclusion:1.The study verified the safety of PBM clinical treatment,and showed that PBM treatment can reduce the inflammatory reaction of patients and promote the recovery of patients’ sensory function.This provides favorable support for the clinical application and promotion of PBM.2.lncRNA TUG1 can be used as a potential target of PBM to regulate the polarization and inflammatory response of macrophages and affect the axon growth of DRG.3.TUG1-miR-1192/TLR3 axis may be the specific mechanism for PBM to play its biological function.4.In vivo treatment of PBM can inhibit the expression of lncRNA TUG1,TLR3 and inflammatory cytokines,and promote the survival of neurons and the recovery of motor function in mice.